Fused hybrid gene

ABSTRACT

This invention is a process to produce specific proteins coded for by eukaryotic (or prokaryotic) DNA in bacteria. The invention, which uses recombinant DNA techniques, produces proteins in their natural, functional state unencumbered by extraneous peptides.

The invention described herein was made in the course of work under agrant or award from the National Science Foundation.

This is a division, of application Ser. No. 111,101, filed Jan. 10,1980, now U.S. Pat. Not. 4,332,892, which is a continuation of Ser. No.3,102 filed Jan. 15, 1979 now abandoned.

This invention is a method for producing in bacteria prokaryoctic oreukaryotic proteins in native, unfused form free from extraneouspeptides.

Recombinant DNA techniques in vitro have been used to insert a varietyof eukaryotic genes into plasmids carried by Escherichia coli in aneffort to induce these bacteria to produce eukaryotic proteins. Most ofthese genes have not directed the synthesis of the native proteinsbecause the eukaryotic signals coding for initiation of transcriptionand/or translation do not function well in E. coli. One proposedsolution to this problem has been the fusion of the eukaryotic gene witha bacterial gene. The process results in the production of a hybridprotein, a portion of which at its carboxyl terminus is constituted bythe eukaryotic protein. In one case, it has been possible to separate asmall biologically active protein from a fusion product (Itakura, K. etal., Science 198, 1956 (1977)).

Gene expression takes place by transcription into mRNA then translationinto protein. To do these operations, the DNA preceding the gene musthave a sequence which: (a) directs efficient binding of bacterial RNApolymerase and efficient initiation of transcription, and (b) codes fora mRNA that directs efficient binding of mRNA to the ribosomes andinitiation of translation into protein.

The present invention provides a method of producing native, unfusedprokaryotic or eukaryotic protein in bacteria which comprises insertinginto a bacterial plasmid a gene for a prokaryotic or eukaryotic proteinand a portable promoter consisting of a DNA fragment containing atranscription initiation site recognized by RNA polymerase andcontaining no protein translational start site, said promoter beinginserted ahead of a protein translational start site of said gene toform a fused gene having a hybrid ribosome binding site, inserting saidplasmid into said bacteria to transform said bacteria with said plasmidcontaining said fused gene, and culturing the transformed bacteria toproduce said prokaryotic or eukaryotic protein.

The present invention utilizes nucleases, restriction enzymes, and DNAligase to position a portable promoter consisting of a DNA fragmentcontaining a transcription site but no translation initiation site nearthe beginning of the gene which codes for the desired protein to form ahybrid ribosomal binding site. The protein produced by the bacteriumfrom this hybrid is the native derivative of the implanted gene. It hasbeen found that the endonuclease digestion product of the E. coli lacoperon, a fragment of DNA which contains a transcription initiation sitebut no translational start site, has the required properties to functionas a portable promoter in the present invention, being transcribed athigh efficiency by bacterial RNA polymerase. The mRNA produced containsa (Shine-Dalgarno (S-D) Sequence) but it does not include the AUG or CUGrequired for translational initiation. However, in accordance with thepresent invention, a hybrid ribosomol binding site is formed consistingof the S-D sequence and initiator from the lac operon and the ATGsequence of the gene, and such a fused gene is translated andtranscribed efficiently. Using the enzymes exonuclease III and S1, thepromoter may be put at any desired position in front of thetranslational start site of the gene in order to obtain optimumproduction of protein. Since the promoter can be inserted at arestriction site ahead of the translational start site of the gene, thegene can first be cut at the restriction site, the desired number ofbase pairs and any single standard tails can be removed by treating withnucleases for the appropriate time period, and religating.

The following specific example is intended to illustrate more fully thenature of the present invention without acting as a limitation upon itsscope.

EXAMPLE

A rabbit β-globin gene was first cloned into the Hin III site of pBR322,a plasmid of the E. coli bacteria, via restriction enzyme cuts of theinitial DNA, reconstitution of the gene by T4 ligase, insertion of thereconstituted gene into the Hin III site using chemically synthesizedHin III linkers, and religating with DNA ligase.

The Hin III cut at the carboxyl end of the cloned gene was removed bypartially digesting with Hin III, filling in the resulting Hin III"sticky ends" with E. coli DNA polymerase I, and religating with T4ligase. This left in the resulting plasmid a single Hin III cut 25 basepairs ahead of the amino terminus of the globin gene.

Differing numbers of the 25 base pairs between the Hin III cut and theATG signalling the start point of translation were removed fromdifferent samples of the cloned gene as follows: the plasmid was cutwith Hin III, resected for various times from 0.5 to 10 minutes with ExoIII, then treated with S1 to remove single-standard tails.

The portable promoter of the lac operon, an R1-Alu restriction fragmentof E. coli DNA, was then inserted by treating each sample of the plasmidwith R1 which cuts at a unique site some 30 base pairs upstream from theHin III site, and the portable promoter was inserted into the plasmidbackbone at this site. This requires one "sticky end" and one "flush"end, both of which are ligated by the same treatment with ligase.

Colonies of E. coli each containing one of these resulting plasmids werethen screened for β-globin production using RIA-screening techniques toidentify the one or more producing β-globin.

The globin gene in the above construction can be any gene coding forprokaryotic or eukaryotic proteins, and any other unique restrictionsite can be employed in place of the Hin III site. If the restrictionsite is located inconveniently far from the beginning of the gene, itmay be moved (for example, a Hin III site may be moved by opening theplasmid with Hin III, digesting with Exo III and S1, then religating theresulting plasmid in the presence of excess Hin III linkers). Anysuitable restriction site can be employed for insertion of the portablepromoter in place of the R1 site (e.g. Pst, BAM, or Sal I). Finally, itshould be emphasized that the most difficult step, the cloning of thegene into the plasmid, is done once and left unchanged. The promoterfragment will confer its constitutive expression on the cell so it iseasy to screen for the intact promoters.

What is claimed is:
 1. A fused hybrid gene capable of expressing nativeunfused prokaryotic or eukaryotic protein consisting essentially of (1)a portable promoter including a portion of a bacterial gene having aShine-Dalgarno sequence and a transcription initiation site recognizedby RNA polymerase and containing no protein translational start site,and (2) fused thereto a gene for a native unfused prokaryotic oreukaryotic protein including its translational start site, said portablepromoter being located upstream from said translational start site.
 2. Afused gene as claimed in claim 1 in which said promoter is the productof restriction endonuclease digestion of an operon.
 3. A fused gene asclaimed in claim 2 in which said bacterial gene is that of E. coli andsaid promoter is the product of restriction endonuclease digestion ofthe lac operon of E. coli.
 4. A fused gene as claimed in claim 1 whereinsaid translational start site of said gene for said prokaryotic oreukaryotic protein is the sequence ATG.
 5. A fused gene as claimed inclaim 1 wherein said portable promoter and said translational start siteof said prokaryotic or eukaryotic gene together comprise a hybridribosomal binding site.
 6. A fused gene as claimed in claim 1 whereinsaid gene for a prokaryotic or eukaryotic protein is a gene for aeukaryotic protein.
 7. A bacterium containing the fused gene of claim 1,said bacterium being capable of producing said prokaryotic or saideukaryotic protein.
 8. A bacterium containing the fused gene of claim 6,said bacterium being capable of producing said eukaryotic protein.